One of our key research goals is to elucidate the molecular mechanisms involved in assembly and function of translation initiation complexes involved in protein synthesis in living cells. We use budding yeast as a model system owing to the powerful combination of genetics and biochemistry that can be employed to dissect complex pathways in vivo. The translation initiation pathway produces an 80S ribosome bound to mRNA with methionyl initiator tRNAMet (Met-tRNAi) base-paired to the AUG start codon. Met-tRNAi is recruited first to the 40S subunit in a ternary complex (TC) with GTP-bound eIF2, to produce the 43S preinitiation complex (PIC), in a manner stimulated by eIFs -1, -1A, and -3. The 43S PIC attaches to the 5 end of mRNA, in a manner facilitated by cap-binding complex eIF4F (comprised of eIF4E, eIF4G, and RNA helicase eIF4A) and PABP bound to the poly(A) tail, and scans the 5 untranslated region (UTR) for the AUG start codon. Scanning is promoted by eIFs -1 and -1A, which induce an open conformation of the 40S, and by eIF4F and other RNA helicases that remove secondary structure in the 5 UTR. AUG recognition by Met-tRNAi evokes irreversible hydrolysis of the GTP bound to eIF2, dependent on GTPase activating protein (GAP) eIF5, releasing eIF2-GDP from the PIC and Met-tRNAi into the ribosomal P site. After joining of the 60S subunit, producing the completed 80S initiation complex, the eIF2-GDP is recycled to eIF2-GTP by guanine nucleotide exchange factor (GEF) eIF2B to prepare for the next round of translation initiation. Mechanism and regulation of the GEF function of eIF2B. The GEF function of eIF2B is down-regulated in amino acid-starved cells by phosphorylation of the a subunit of eIF2 by protein kinase Gcn2, provoking a reduction in general protein synthesis. The eIF2B contains five subunits, but the e/Gcd6 subunit is sufficient for GEF activity in vitro. The d/Gcd2, b/Gcd7 and a/Gcn3 subunits are related in sequence and comprise a regulatory subcomplex in eIF2B that mediates tight binding of phosphorylated eIF2(aP)-GDP, via the S1 domain in eIF2a, which is required to inhibit GEF function. Regulation of eIF2B by eIF2(aP) is not essential in nutrient-replete cells, but the eIF2B regulatory subunits d/Gcd2 and b/Gcd7 are required for cell viability, and their indispensable functions are not well understood. We showed recently that (i) depletion of b/Gcd7 from cells, (ii) three lethal b/Gcd7 amino acid substitutions, and (iii) a synthetically lethal combination of substitutions in b/Gcd7 and eIF2a all impaired eIF2 binding to eIF2B, without reducing e/Gcd6 abundance in eIF2B. These and other findings provided strong evidence that b/Gcd7 is essential for binding the eIF2B substrate (unphosphorylated eIF2-GDP), beyond its regulatory role in mediating eIF2B inhibition by phosphorylated eIF2. Roles of eIF4G in mRNA activation and determining translational efficiencies genome-wide. Attachment of the 43S PIC near the 5 end of mRNA is enhanced by binding of eIF4F (eIF4E, eIF4G, eIF4A) to the cap structure, PABP binding to the poly(A) tail, and PABP-eIF4G interaction, which produces a closed loop mRNP thought to be crucial for translation initiation. Budding yeast contains two functionally interchangeable eIF4G isoforms (encoded by TIF4631 and TIF4632). Key evidence that PABP-eIF4G interaction is important in vivo came originally from the finding that, in yeast cells lacking eIF4G2, a temperature-sensitive mutation in the eIF4E-binding site of eIF4G1 (tif4631-459) is synthetically lethal with an N-terminal truncation that eliminates the PABP-binding domain of eIF4G1 (-delN300). However, we found that an internal deletion of the PABP binding domain of tif4631-459 is not lethal, and that the RNA-binding domain at the extreme N-terminus (RNA1) must be removed simultaneously to produce a lethal reduction in eIF4G1-459 function. We further showed that the region between RNA1 and the PABP binding domain contains two segments conserved in yeast eIF4G homologs, Box1 and Box2, that also functionally overlap with the PABP-binding domain in vivo. We obtained biochemical evidence that RNA1, Box1, Box2, and the PABP-binding domain all contribute to assembly of native eIF4G&#8729;mRNA&#8729;PABP complexes, quantified by assaying mRNA-dependent (RNAse-sensitive) coimmunoprecipitation of PABP with eIF4G from cell extracts. In collaboration with Jon Lorschs group at Johns Hopkins Medical School, we found that RNA1 and Box1 both enhance direct PABP binding to the eIF4G1 NTD, and that Box1 contributes to RNA binding by the eIF4G1 NTD in vitro. Thus, while the eIF4G-PABP interaction is important, RNA binding by the eIF4G1 NTD makes an overlapping contribution to mRNA activation in vivo, and multiple segments in the NTD contribute to the PABP and RNA binding activities of the eIF4G1 NTD. Our results are significant in demonstrating that closed-loop mRNP formation via PABP-eIF4G interaction is nonessential in vivo. We propose that the eIF4G-PABP interaction simply represents one of several interactions that stabilize eIF4G binding to mRNA, which is likely the critical event in promoting 43S attachment. In addition to eIF4F's role in promoting 43S attachment to mRNA, the helicase activity of eIF4A likely facilitates scanning through secondary structure in the 5 UTR, and eIF4F is expected to determine translational efficiencies (TEs) among different mRNAs. We tested this hypothesis by measuring the effect of depleting eIF4G in vivo on TEs genome-wide, using cDNA microarrays to quantify the ratio of mRNA abundance in polysomes versus total RNA. Both eIF4G isoforms were eliminated in a strain lacking TIF4632 and harboring a tif4631-td degron allele, which enables transcriptional repression and rapid degradation of eIF4G1 in non-permissive conditions. The elimination of eIF4G reduced protein synthesis rates 3-fold and arrested cell growth but, surprisingly, the TEs of most mRNAs were not greatly affected. An intriguing consequence of eIF4G depletion, however, was to narrow the range of TE values by reducing translation of many mRNAs that exhibit higher than average TEs in WT, while increasing translation of other mRNAs with lower than average TE values. Thus, while eIF4G is not essential for translation of any mRNAs in yeast, it is an important rate-enhancing factor and also promotes the differentiation of TEs among mRNAs to an extent that is essential for cell division. An Upstream ORF with non-AUG Start Codon is Translated in vivo but Dispensable for Translational Control of GCN4 mRNA Genome-wide analysis of ribosome locations in mRNAs of Saccharomyces cerevisisae revealed a high level of translation of upstream open reading frames (uORFs) that initiate with non-AUG start codons in many transcripts. Two such non-AUG-initiated uORFs (nAuORFs 1 and 2) occur in GCN4 mRNA upstream of the four AUG-initiated uORFs (uORFs 1 to 4) that mediate translational control of GCN4 mRNA. We have tested the proposal that these nAuORFs play an important role in this regulatory mechanism. We verified that nAuORF2 is translated in vivo by demonstrating b-galactosidase production from lacZ coding sequences fused to nAuORF2, in a manner abolished by replacing its AUA start codon with the non-cognate triplet AAA;however, translation of nAuORF1 was not detected. Importantly, replacing the near-cognate start codons of both nAuORFs with non-cognate triplets had little or no effect on the repression of GCN4 translation in nutrient-replete cells, nor on its derepression in response to histidine limitation or other stress conditions. Additionally, we found no evidence that initiation from the AUA codon of nAuORF2 is substantially elevated, nor dependent on Gcn2, in histidine-deprived cells. Thus, while nAuORF2 is translated, this event is not critical for GCN4 translational control.